Mitochondrial dysfunctions cause a variety of neurodegenerative and age-associated diseases. The mechanisms of pathogenesis of human diseases due to mitochondrial dysfunction are poorly understood. It is expected that apart from the structural-functional defects in respiratory complexes, deficiencies in their regulatory mechanisms could also have important implications in pathophysiology. Since cAMP signaling plays important role in both neuronal plasticity and survival, it may also control the mitochondrial respiratory chain, which apart from its bioenergetic functions plays important roles in neuronal physiology by regulating intracellular Ca2+, and superoxide (O2.-) production. We base our hypothesis on the following observations: 1) cAMP-dependent phosphorylation of respiratory complexes in in vitro experiments with isolated mitochondria [1-4], and 2) reduced cellular respiration in cortical neurons grown in physiologically normoxic conditions (3% oxygen) upon treatments with cAMP agonists (this study). Based on these observations, the goal of this proposal is to investigate the regulation of mitochondrial respiratory chain function in intact neurons by cAMP under physiological conditions, and to identify the molecular targets of regulation. The specific aims are: I. Characterization of cAMP-mediated regulation of respiratory chain function: Specifically, we will study the effects of intracellular cAMP elevation on mitochondrial respiration and superoxide (O2.-) production from the respiratory chain in primary neurons using two recently developed techniques from this group, in situ respirometry and confocal imaging of mitochondrial superoxide (O2.-) production. II. Identification of the molecular targets of cAMP-mediated regulation within the respiratory chain: Respiratory complex I is one of the expected molecular targets of cAMP mediated regulation. We will determine the phosphorylation states of complex I subunits by 2-dimensional blue-native (BN-) /SDS-PAGE and mass-spectrometry, and study the role of selected subunits on complex I function using molecular genetic approaches. A successful outcome to our proposed study will for the first time establish the reversible regulation of the respiratory chain by cAMP in intact neurons, and provide an experimental system to study the relevance of cAMP-mediated regulation of the respiratory chain in the pathophysiology of neurodegenerative and age-related disorders. PUBLIC HEALTH RELEVANCE: Mitochondrial dysfunction is associated with a large number of neuromuscular diseases with onset in childhood to later in life. We expect this study will lead to a better understanding of the pathological mechanisms of neurodegenerative diseases associated with mitochondrial dysfunction.